Insulin resistance stands as a significant threat to public health worldwide, and a largely unmet medical need. To unravel the complex biology of this protean syndrome, we have endeavored to apply genetic techniques to probe gene function and tissue interactions related to metabolism, and identify tractable targets for pharmacological intervention in type 2 diabetes. Over the past five years, the notable contributions of this grant to our knowledge of the insulin resistance syndrome have been: (i) a critical reassessment of the relative roles of insulin- dependent and -independent mechanisms of glucose disposal in metabolic homeostasis; (ii) the discovery of mechanisms by which non-canonical sites of insulin action, such as central nervous system (CNS) and endocrine pancreas, play an early and decisive role in the progression from insulin resistance to diabetes; and (iii) the notion that pancreatic beta cells are an insulin-sensitive cell type. Building on these lessons, the PI proposes studies of the integrated physiology of insulin action that will focus primarily on the contribution of CNS, liver and enteroendocrine system to the insulin-resistant state. The PI presents data putatively identifying an insulin-sensitive neuron population, characterized by Glut4 expression; as well as a novel gut epithelial progenitor cell with the unique ability to give rise to bona fide insulin-secreting, Beta-like endocrine cells in vivo. Three aims are outlined: Aim 1 will tackle the role of insulin signaling in brain glucose metabolism, using a novel approach to identify Glut4-expressing neurons and characterize their contribution to systemic metabolism. Aim 2 will delve into the pathophysiology of hepatic insulin resistance, and specifically into the identification of transcription factors that cause the paradoxical admixture of increased glucose production and triglyceride synthesis that characterizes the diabetic liver. Aim 3 will study the role of insulin signaling in a newly identified sub-population of gut epithelial progenitor cells that express the insulin-regulated transcription factor Foxo1 and show the surprising ability to be reprogrammed into Beta-like cells that secrete insulin in response to glucose. The proposed body of work will advance our understanding of the insulin-resistant syndrome at the biochemical, genetic, and integrated physiological levels, with the ultimate goal of translating newly acquired information into innovative approaches to its treatment. PUBLIC HEALTH RELEVANCE: In the last decade, we endeavored to unravel the 'insulin code'-a concerted succession of biochemical and cell biological events that underlie the integrated physiology of insulin action. We documented the contribution of Insulin Receptor, its main substrates and signaling intermediates to the key abnormalities of insulin resistance: decreased glucose disposal, increased glucose production, and impaired pancreatic beta cell function. We used genetic techniques to probe hormonal and nutritional communication among different tissues, and analyze the progression from insulin resistance to overt diabetes. We demonstrated how tissues with insulin-dependent glucose disposal (muscle and fat) interact with those that utilize glucose independent of insulin (liver, brain, pancreatic islets) to determine various aspects of insulin resistance. We developed a theory integrating 2 cell function with insulin resistance; and delved into the pathophysiology of the brain's contribution to glucose homeostasis. We also examined the bimodal involvement of the liver in diabetes as a site of mixed sensitivity and resistance to insulin. In the process, we burrowed into the Foxo1 pathway, demonstrating the central position of this transcription factor in the regulation of hepatic glucose production and neuropeptide processing. Reversal of insulin resistance remains a critical goal of diabetes treatment, and arguably one of the largest unmet medical needs. Work supported by this grant has defined mechanisms that we believe to be new, linking insulin action in specific organs and cell types with the pathophysiology of insulin resistance, and disclosing biochemical and molecular circuits that can be exploited for diabetes treatment. We envision that the studies proposed for the next funding cycle will reveal new dimensions to the insulin resistance syndrome and expand the repertoire of currently available targets for diabetes therapy and prevention. The proposed experiments build on lessons of the past funding cycle to explore the contribution of newly identified mechanisms to metabolic homeostasis. The driving theme of our work is to define molecular pathways that can be enlisted in the clinic against insulin resistance.